Female gonads house a finite number of meiotically-arrested germ cells (oocytes) enclosed within primordial follicles that serve as the stockpile of eggs released at ovulation at each menstrual cycle for potential fertilization. Gougeon, Endocr Rev. 17, 121 (1996); Morita & Tilly, Dev. Biol. 213 (1999). Once depleted, the ovarian germ cell pool cannot be replenished. Thus, exposure of women to a wide spectrum of agents that damage the ovary, such as chemotherapeutic agents and radiotherapy, generally leads to premature menopause and irreversible sterility. Waxman, Soc. Med. 76, 144 (1983); Familiari et al., Hum. Reprod. 8, 2080 (1993); Ried & Jaffe, Semin. Roentgenol. 29, 6 (1994); and Reichman & Green, Monogr. Natl. Cancer Inst. 16, 125 (1994).
Apoptotic cell death plays a fundamental role in normal germ cell endowment and follicular dynamics in the ovary. Tilly & Ratts, Contemp. Obstet. Gynecol. 41, 59 (1996); Tilly, Rev. Reprod. 1, 162 (1996); and Tillyet al., Cell Death Differ. 4, 180(1997). Cell fate in the ovary is likely dependent on the actions of several proteins recently identified as key determinants of cell survival or death (Adams & Cory, Science 281,1322 (1998); Green, Cell 94,695 (1998); Thornberry & Lazebnik, Science 281,1312 (1998); Reed, Oncogene 17,3225 (1998); Korsmeyer, Cancer Res. 59,1693 (1999). Among these identified in the ovary are p53 (Tilly et al., Endocrinology 136, 1394 (1995); Keren-Tal et al., Exp. Cell Res. 218, 283 (1995); and Makrigiannakis et al., J. Clin. Endocrinol. Metab. 85,449 (2000)), members of the bc1-2 gene family (Tilly et al., Endocrinology 136-232 (1995); Ratts et al., Endocrinology 136,3665 (1995); Knudson et al., Science 270,99 (1995); Perez et al., Nature Med. 3 1228 (1997); Kugu et al., Cell Death Differ. 5, 67 (1998); Perez et al., Nature Genet. 21, 200 (1999), and members of the caspase gene family (Flaws et al., Endocrinology 136, 5042 (1995); Perez et al., Nature Med. 3, 1228 (1997); Maravei et al., Cell Death Differ. 4, 707 (1997); Kugu et al., Cell Death Differ. 5, 67 (1998); Boone & Tsang, Biol. Reprod. 58, 1533 (1998); Bergeron et al., Genes Dev. 13, 1304 (1998); and Perez et al., Mol. Hum Reprod. 5, 414 (1999)).
In addition, ceramide, a recently identified lipid second messenger associated with cell death signaling (Spiegel et al., Curr. Opin. Cell Biol. 8, 159 (1996); Hannun, Science 274, 1855(1996); and Kolesnick & Kronke,Annu. Rev. Physiol. 60,643 (1998)) has been implicated in the induction of apoptosis in the ovary (Witty et al., Endocrinology 137, 5269 (1996); Kaipia et al., Endocrinology 137,4864(1996); and Martimbeau & Tilly, Clin. Endocrinol. 46, 241(1997)).
Since the initial discovery ofthe sphingomyelin pathway, numerous studies have been published on the potential role of ceramide in signaling cell death (Hannun, (1996) id.; and Kolesnick & Kronke (1998) id.). A central role for ceramide, a pro-apoptotic sphingolipid (Kolesnick & Krönke, Annu. Rev. Physiol., 60:643 (1998)) derived from either sphingomyelin hydrolysis or de novo synthesis, in mediating death of oocytes exposed to anti-cancer therapies, has recently emerged (Perez et al., Nat. Med., 3:1228 (1997); and Morita, Y. et al.,Nat. Med. 6, 1109-1114 (2000)). Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy (Morita, Y. et al.,Nat. Med. 6, 1109-1114 (2000)). Whether or not cells die in response to ceramide elevations is, however, at least partly dependent upon the rate at which ceramide is metabolized. It is now known that ceramide can also be metabolized via ceramidase to sphingosine, which is then phosphorylated by sphingosine kinase to generate sphingosine-1-phosphate (S1P), a potent antagonist of ceramide-promoted apoptosis (Cuvillier et al., Nature 381, 800 (1996); Spiegel et al., Ann. N.Y. Acad. Sci 845, 11 (1998); and Spiegel, J. Leukoc. Biol. 65, 341 (1999)).
In some cell types, S1P can effectively counterbalance stress-kinase activation and apoptosis induced by membrane-permeant ceramide analogs or external stressors known to work through elevations in intracellular ceramide levels. Therefore, a rheostat model has been proposed in which cell fate is controlled by shifts in the balance between ceramide and S1P levels. However, the physiologic importance of ceramide, and that of sphingomyelin hydrolysis as a whole, in activating developmental or homoeostatic paradigms of apoptosis have recently been questioned by some investigators (Hofmann & Dixit, Trends Biochem. Sci 23, 374 (1998); and Watts et al., Cell Death Differ. 6, 105 (1999)). In particular, Hofmann et al., describe a lack of developmental defects that should be the consequence of impaired apoptosis in the acid sphingomyelinase (ASMase) gene knockout mouse as substantive evidence against a role for ASMase-catalyzed sphingomyelin hydrolysis and ceramide in signaling cell death (Kolesnick & Kronke (1998) id.)
Earlier studies using pharmacologic and genetic approaches have shown that several other components of the programmed cell death regulatory pathway in oocytes, including Bc1-2 family members (Ratts et al., Endocrinology 136, 3665 (1995); Perez et al., Nat. Med. 3, 1228 (1997); Morita et al., Mol. Endocrinol. 13, 841 (1999); Perez et al., Nat. Genet. 21,200(1999)); and caspases (Perez et al.,(1997) id.; Bergeron et al., Genes Dev. 12, 1304 (1998)), are required for oocyte survival or death. However, cell lineage specificity will certainly be an important issue to consider based on observations that p53, a classic signaling molecule for cancer therapy-induced tumor cell destruction (Ko & Prives, Genes Dev. 10, 1054 (1996); and Ding et al., Crit. Rev. Oncog. 9, 83 (1998)), is completely dispensable for oocyte death initiated by cancer therapy (Perez et al., (1997) id.)
Although the sensitivity of oocytes to cancer therapy, and the potential role of ceramide in signaling cell death are reported, as evidenced above, little is known regarding the mechanisms responsible for female germ cell destruction. Recently, it has been shown that female mouse oocytes undergo a type of cell death, referred to as apoptosis, when exposed in vitro to a prototypical anti-cancer drug (doxorubicin, 14-hydroxydaunorubicin, Adriamycin®). (Perez et al., (1997) id.) Moreover, it was shown that culture of mouse oocytes in vitro with sphingosine-1 -phosphate protected the oocytes from death induced by subsequent doxorubicin exposure for up to 24 hours. However, the protection was only tested in vitro with only a single drug under a brief window of time, and thus in vivo application remained questionable. Also, the oocytes isolated for these in vitro tests are developmentally very different from the specific populations of oocytes that are destroyed by chemotherapy and radiotherapy in vivo. Due to the differences in oocytes, it is impossible to determine the relevance of data derived from these in vitro models to that which occurs in vivo. Thus, there remains a need for in vivo methods of protecting the female reproductive system from natural or artificial insult.